DNA-seq FAQs

The PicoPLEX Gold Single Cell DNA-Seq Kit is used to amplify genomic DNA from single cells to reliably detect chromosomal aneuploidies, copy number variations (CNVs), and single nucleotide variants (CNVs) using next-generation sequencing on Illumina platforms.

Applications include preimplantation genetic testing (PGT) using blastomeres and trophectoderm cells as well as analysis of chromosomal abnormalities and mutations in circulating tumor cells for cancer research.

No. The PicoPLEX Gold Single Cell DNA-Seq Kit offers robust and reproducible amplification of DNA from single cells for the detection of copy number variations (CNVs), single nucleotide variants (SNVs), and other chromosomal anomalies. This kit should not be used for high-coverage deep sequencing such as de novo sequencing and/or complete (whole genome) resequencing.

The PicoPLEX Gold Single Cell DNA-Seq Kit lyses cells and prepares appropriately-barcoded NGS-ready libraries in less than 3 hours.

Single blastomeres, trophectoderm cells, and cultured cells have been used successfully.

Yes, cells stained with surface antibodies can be used if the cells are not fixed.

Yes.

Our DNA single index, unique dual index, and HT dual index kits are compatible with the PicoPLEX Gold Single Cell DNA-Seq Kit.

No. Only DNA single index, unique dual index, and HT dual index kits have been approved for use with the PicoPLEX Gold Single Cell DNA-Seq Kit.

The DNA single index, unique dual index, and HT dual index kits are compatible with the PicoPLEX Gold Single Cell DNA-Seq Kit.

The first 11 cycles of each read will contain quasi-random bases introduced during the PicoPLEX Gold Single Cell DNA-Seq library preparation. For sequence alignment, either trim the initial 14 bases from each read or begin calibration and data collection at base position 15.

No, this kit is not compatible with Ion Torrent or Pac Bio.

Libraries prepared using the PicoPLEX Gold Single Cell DNA-Seq Kit are compatible with both single- and paired-end sequencing on Illumina platforms.

Libraries prepared using the PicoPLEX Gold Single Cell DNA-Seq Kit result in a broad size-range distribution of fragments, typically ranging from ~300–1,000 bp total size (~200–900 bp insert size).

The PicoPLEX Gold Single Cell DNA-Seq Kit must be run on a thermal cycler that can accommodate 50-µl reaction volumes and that is equipped with a heated lid. Set the temperature of the heated lid to 100–105°C to avoid sample evaporation during incubation and cycling.

A real-time PCR cycler is recommended to monitor amplification by the addition of fluorescent dye (not provided with the kit) to the reaction. If a regular thermal cycler is used instead, there is no need to add the dye. Substitute with an appropriate amount of nuclease-free water to adjust volume while preparing the Amplification Reaction Master Mix. In the absence of a real-time thermal cycler, use a regular thermal cycler and quantify library amplification via gel or Bioanalyzer analysis.

EvaGreen dye (Cat. No. 31000-T, Biotium Inc.) is recommended due to its high sensitivity and low interference with amplification chemistry. Depending on the real-time instrument used, a calibration dye may also be needed. Please refer to your real-time PCR instrument's user manual for additional information.

Quantify PicoPLEX Gold Single Cell DNA-Seq libraries using real-time qPCR using the appropriate instrument-specific Illumina library quantification kit. Pooled, purified libraries are diluted 50,000–500,000-fold and used as the template for quantification by real-time qPCR. It is recommended to use 300 bp as the size for calculating the library concentration.

With libraries made from a single cell using the PicoPLEX Gold Single Cell DNA-Seq Kit, a good starting point is to consider a library size of 300 bp and load 16 pM on the MiSeq® v3. Other platforms will have different loading concentrations, so we recommend following the manufacturers' instructions. It is very important to add at least 5% PhiX DNA to the library prior to loading on the flow cell to achieve optimal diversity.

Mix the beads and the sample(s) at a 1:1 ratio for most NGS-based CNV detection purposes.

More than 90% of the reads from libraries generated with the PicoPLEX Gold Single Cell DNA-Seq Kit fall between 25% and 70% GC content.

The greatest advantage of PicoPLEX over other WGA technologies is the reproducibility obtained from cell to cell while using single cells. This technology also yields a very low background.

• 1–10 human cells (e.g., blastomeres, polar bodies, trophoblasts, amniocytes, CTCs, or cultured cells)
• 1,000–10,000 bacterial cells
• Intact or fragmented, single- or double-stranded DNA
• Isolated DNA (15–60 pg of human DNA)

  NOTE: Inputs higher than 60 pg are possible, but require cycle optimization at certain steps

• Input size over 500 bases
• Sorted chromosomes

Single blastomeres, polar bodies, trophoblasts, amniocytes, and cultured cells have been amplified successfully.

Yes. Cells should be washed to minimize extracellular DNA or growth media contaminants. We recommend washing in PBS (Ca²⁺-free, Mg²⁺-free, and BSA-free) and limiting carryover of wash buffer to less than 2.5 μl.

While this has not been validated at Takara Bio, some customers have reported successfully using polyvinyl alcohol (0.1% PVA, for example) in PBS to wash/collect their cells. Additionally, your washing/collecting buffer (PBS) must be Ca²⁺- and Mg²⁺-free.

Flow sorting, dilution, and micromanipulation are compatible with PicoPLEX NGS kits.

We strongly recommend using light scattering or phase contrast, and not fixing, to sort or collect samples. Microscopic/visual confirmation of successfully sorted cells can be used to optimize sorting conditions.

The kit will accommodate a 5-μl sample input volume per reaction, with a maximum of 2.5 μl of cell and buffer carryover.

No, the PicoPLEX Single Cell WGA Kit uses different technology.

The PicoPLEX Single Cell WGA Kit lyses cells and amplifies to >5 μg of end product DNA in <3 hours.

The PicoPLEX Single Cell WGA Kit has about a 90% amplification success rate with flow-sorted tissue culture cells, limited by the uncertainties of sorting rather than by amplification. Single cells always give a high, reproducible yield of amplified genomic DNA. Buffer controls should always give virtually no background.

Yes, with about a 90% correlation coefficient for qPCR Ct data from replicate single-cell reactions.

See the table below for third-party data obtained using the PicoPLEX Single Cell WGA Kit.

Yes. The PicoPLEX Single Cell WGA Kit amplifies with single-copy sensitivity and high specificity.

The end product of the PicoPLEX Single Cell WGA Kit is a mixture of single- and double-stranded DNA.

The PicoPLEX Single Cell WGA Kit can be used for:

• Copy number variation (CNV) analysis: superior for oligonucleotide and array CGH
• SNP genotyping
• Mutation detection by PCR

The PicoPLEX Single Cell WGA Kit particularly excels at copy number variation analysis.

The PicoPLEX Single Cell WGA Kit is well suited for SNP genotyping because the same loci are reproducibly amplified between different cells. SNPs contained within well-represented regions can be accurately detected without the stochastic dropouts and noise associated with other WGA methods.

The PicoPLEX Single Cell WGA Kit has not been optimized for gel-based STR genotyping.

Yes, as long as the amplimers are less than 250 bp.

Random-prime labeling and chemical labeling.

For microarrays, we recommend starting with the amount recommended in the instructions from the assay manufacturer and titrating if necessary to improve results even further.

For PCR assays, we recommend using 5–200 ng of amplified PicoPLEX Single Cell WGA Kit library.

No. The PicoPLEX Single Cell WGA Kit does not have adapters nor barcodes. If NGS is the desired application, the WGA material can be used in a library preparation technology (e.g., the ThruPLEX DNA-Seq Kit).

Takara Bio does not support this application, but please check the following references for recommended DNA fragmentation sizes:

Yes. PicoPLEX Gold DNA-Seq Kits can be used with Illumina NGS platforms.

The ThruPLEX DNA-Seq Kit is the fastest and most sensitive library prep kit available. Repair, ligation, and amplification are performed in a single tube in three simple steps.

• Input amount: 50 pg to 50 ng
• No cleanup steps
• Number of steps to library: 3
• Time to library: less than 2 hours
• Number of sample transfers: 0

Yes, the ThruPLEX DNA-Seq Kit produces uniform coverage in GC-rich regions.

No, DNA must be double-stranded for use with the ThruPLEX DNA-Seq Kit.

Yes. If the DNA is >1 kb in size, it must be fragmented prior to use with the ThruPLEX DNA-Seq Kit.

DNA fragmentation can be accomplished either mechanically (e.g., Covaris, Bioruptor) or enzymatically (e.g., our DNA Fragmentation Kit).

Successful shearing has been accomplished with 2–50 ng of DNA in 50–130 µl of TE or low-TE buffer. If you are working with smaller amounts of DNA, see the "Low-volume DNA shearing for ThruPLEX library prep" tech note.

Covaris-recommended settings should be used to obtain the desired template fragment size. Please refer to the Covaris website for additional guidance.

Our DNA Fragmentation Kit can be used to prepare randomly fragmented genomic DNA for use with the ThruPLEX DNA-Seq Kit by following the instructions in the DNA Fragmentation Kit User Manual.

The ThruPLEX DNA-Seq Kit has been designed to amplify an input range of 50 pg to 50 ng. The input amount depends on the desired level of complexity needed and the application.

Yes, it necessary to use indexing reagents, which must be purchased separately. They are supplied in tubes or a microplate and consist of amplification primers containing Illumina-compatible indexes, which must be used in the ThruPLEX DNA-Seq Library Amplification Reaction.

Please refer to the ThruPLEX DNA-Seq Kit User Manual for information on selecting the optimal number of cycles for library amplification.

Yes, the ThruPLEX DNA-Seq Kit can be used even if a lab does not have access to a real-time PCR machine. Please refer to the ThruPLEX DNA-Seq Kit User Manual, which contains a table that provides a guide for selecting the number of PCR cycles based on the DNA input amount used.

Yes, ThruPLEX DNA-Seq libraries can be further amplified without adding extra reagents after storage (4°C for up to 6 hours, or –20°C for up to 7 days). Please refer to the ThruPLEX DNA-Seq Kit User Manual for the library processing workflow and instructions on performing additional amplification cycles.

No.

The amount of amplified library can range from 100 ng to 1 µg depending upon many variables, including sample type, fragmentation size, and thermal cycler used. When starting with Covaris-fragmented reference DNA with an average size of 200 bp (using the recommended number of amplification cycles in the ThruPLEX DNA-Seq Kit User Manual), typical yields range from 300 to 700 ng.

Quantification must be performed prior to sequencing. It is normally done after the AMPure XP purification step, but libraries can also be quantified immediately following the Library Amplification Reaction to normalize samples prior to pooling. If yield is of concern, the quantification method used must not require purification of the library. Please refer to the ThruPLEX DNA-Seq Kit User Manual for the library processing workflow after library preparation and for instructions on library quantification.

No, it is only necessary to quantify your individual or pooled libraries prior to sequencing. If you choose to quantify your libraries after the Library Amplification Reaction, it is recommended to do a final quantification of the purified libraries prior to sequencing because AMPure XP purification can result in loss of DNA.

Yes, we suggest using a 1:1 bead to sample ratio. Additionally, a freshly prepared solution of 80% must be used in all washing steps of the protocol. Please refer to the ThruPLEX DNA-Seq Kit User Manual for detailed instructions on AMPure XP purification of ThruPLEX DNA-Seq libraries for next-generation sequencing.

Libraries prepared with the ThruPLEX DNA-Seq Kit are compatible with all Illumina sequencing platforms, including the HiSeq®, MiSeq®, NextSeq® 500, GAIIx™, NovaSeq™, and MiniSeq™.

Please follow Illumina’s recommendations for the optimal loading concentration specific to the version of flow cell you are using. For sequencing on the Illumina MiSeq®, v3, we suggest that you load 14–15 pM of ThruPLEX DNA-Seq library.

No, there are no special steps. ThruPLEX DNA-Seq libraries should be handled and processed in the same way as libraries generated using Illumina library preparation kits, such as the TruSeq® Nano DNA HT Sample Prep Kit.

Follow the manufacturer's instructions for using PhiX. For low-diversity libraries or if one is experiencing sequencing issues, the PhiX concentration may need to be increased. Please refer to the ThruPLEX DNA-Seq Kit User Manual for additional sequencing recommendations.